Intracellular delivery vehicles

ABSTRACT

The invention provides methods and compositions relating to intracellular delivering of agents to eukaryotic cells. The compositions include microbial delivery vehicles such as nonvirulent bacteria comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent. The foreign agent may be a nucleic acid or protein, and is frequently bioactive in and therapeutic to the target eukaryote. In addition, the invention provides eukaryotic cells comprising the subject nonvirulent bacteria and nonhuman eukaryotic host organisms comprising such cells.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuing application under 35 U.S.C. §120, of U.S. Ser. No.09/469,197, filed Dec. 21, 1999, now U.S. Pat. No. 6,287,556, which is acontinuing application of U.S. Ser. No. 09/133,914, filed Aug. 13, 1998,now U.S. Pat. No. 6,004,815, which are incorporated herein by reference.

The disclosed inventions were made with Government support under Grant(Contract) Nos. A127655-10 BD37 awarded by the National Institutes ofHealth. The government may have rights in these inventions.

INTRODUCTION

1. Field of the Invention

The field of this invention is microbial-based intracellular delivery ofagents to eukaryotic cells.

2. Background

The efficient delivery of macromolecules to the cytosol of mammaliancells is of fundamental importance in such processes as the generationof transfected phenotypes and the study of protein function andlocalization. Furthermore, delivery of macromolecules to the cytosol isalso important for the induction of cell-mediated immunity and is asignificant challenge facing the rational design of vaccines tointracellular pathogens. Numerous methodologies currently exist fordelivering macromolecules to mammalian cells. These include but are notlimited to: mechanical techniques such as electroporation (1) andmicroinjection (2); fusion methodologies such as fusion with vesiclesand liposomes (2); chemical treatments employing the use of ATP or EDTA(3) or the external addition of molecules mixed with pore-forming toxinssuch as α-toxin of Staphylococcus aureus (4). Many of these methods havea disadvantage in that the molecule to be delivered may requirelaborious purification (i.e. protein) or the delivery method is limitedto use in vitro. In order to overcome these obstacles, investigatorshave sought to use biological vectors that can enter tissues, cells andspecific cellular compartments for the delivery of macromolecules. Thesevectors are often derived from either retroviruses or bacteria that haveevolved to invade and replicate in specific hosts, organs, or celltypes. While retroviral vectors have been used for the delivery andsubsequent expression of DNA in host cells (5-7), bacterial vectors havebeen exploited primarily for the delivery of antigenic proteins and morerecently adapted for the delivery of DNA to mammalian cells (8-12).

Relevant Literature

Lee et al. (1997) U.S. Pat. No. 5,643,599 and Lee et al. (1996)J.Biol.Chem. 271, 7249-7252 describe hemolysin loaded liposomes forintracellular delivery of macromolecules. Dietrich, G., et al. (1998)Nature Biotech. 16, 138-139 and Ikonomidis, G., et al. (1997) Vaccine15, 433-440 describe the use of Listeria monocytogenes as a macrophagedelivery vehicle. Sizemore, D. R., Branstrom, A. A., & Sadoff, J. C.(1995) Science 270, 299-302 and Courvalin, P., et al. (1995) C. R. Acad.Sci. III 318, 1207-1212 describe the use of attenuated Shigella andinvasive strains of Shigella flexneri and E. coli, respectively, as aDNA delivery vehicle. Hess, J., et al. (1998) Proc. Natl. Acad. Sci.U.S.A. 95, 5299-5304; and Darji, A., et al. (1995) J. Biotechnol. 43,205-212 describe the expression of listeriolysin in several heterologoussystems: an invasive E. coli, Mycobacterium bovis and Listeria innocua,respectively. Moriishi et al. (1996) FEMS Immunol. Med. Microbiol. 16,213-222, 217 describe the transformation of an E.coli with a plasmidencoding listeriolysin. Sanderson, S., Campbell, D. J., & Shastri, N.(1995) J. Exp. Med. 182, 1751-1757, describe the cloning of a Listeriamonocytogenes genomic library in E. coli. Higgins and Portnoy (1998)Nature Biotech. 16, 181-185 review bacterial delivery of DNA.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating tointracellular delivering of agents to eukaryotic cells. The compositionsinclude microbial delivery vehicles such as nonvirulent bacteriacomprising a first gene encoding a nonsecreted foreign cytolysinoperably linked to a heterologous promoter and a second gene encoding adifferent foreign agent. In particular embodiments, the bacteria may bevariously invasive to the target cell, autolysing within target cellendosomes and preferably, a laboratory strain of E. coli. The cytolysinmay lack a functional signal sequence, and is preferably alisteriolysin. The foreign agent may be a nucleic acid or protein, andis frequently bioactive in and therapeutic to the target eukaryote. Inaddition, the invention provides eukaryotic cells comprising the subjectnonvirulent bacteria and nonhuman eukaryotic host organisms comprisingsuch cells. The invention also provides methods for introducing foreignagents into eukaryotic cells comprising the step of contacting the cellin vivo or in vitro with the subject bacteria under conditions wherebythe agent enters the cell. In particular embodiments, the bacterium isendocytosed into a vacuole of the cell, undergoes lysis and thecytolysin mediates transfer of the agent from the vacuole to the cytosolof the cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Presentation of SL8/K^(b) complex to B3Z T-cells.

FIG. 2A and 2B. Time requirement for presentation of SL8/K^(b) complex.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The following preferred embodiments and examples are offered by way ofillustration and not by way of limitation.

The subject bacteria comprise a first gene encoding a nonsecretedforeign cytolysin operably linked to a heterologous promoter. A widevariety of foreign (i.e. not native to the microbial delivery vehicle)cytolysins may be used so long as the cytolysin is not significantlysecreted by the microbe and facilitates cytosolic delivery of theforeign agent as determined by the assays described below. Exemplarycytolysins include phospholipases (see, e.g., Camilli, A., et al., J.Exp. Med. 173:751-754 (1991)), pore-forming toxins (e.g., analpha-toxin), natural cytolysins of gram-positive bacteria, such aslisteriolysin O (LLO, e.g. Mengaud, J., et al., Infect. Immun.56:766-772 (1988) and Portnoy, et al., Infect. Immun. 60:2710-2717(1992)), streptolysin O (SLO, e.g. Palmer M, et al., 1998, Biochemistry37(8):2378-2383) and perfringolysin O (PFO, e.g. Rossjohn J, et al.,Cell 89(5):685-692). Where the target cell is phagosomal, acid activatedcytolysins may be advantageously used. For example, listeriolysin Oexhibits greater pore-forming ability at mildly acidic pH (the pHconditions within the phagosome), thereby facilitating delivery of theliposome contents to the cytoplasm (see, e.g., Portnoy, et al., Infect.Immun. 60:2710-2717 (1992)). Furthermore, natural cytolysins are readilymodified to generate mutants which are screened in the assays describedbelow or otherwise known in the art (e.g. Awad M M, et al., MicrobPathog. 1997, 22(5): 275-284) desired activity modifications. Ingeneral, the screening assays measure the ability of a candidatecytolysin to confer on a bacterium the ability to render a target cellvacuole permeable to a label (e.g., a fluorescent or radioactive label)that is contained in the vacuole. In a particular example, the inventionprovides mutations in natural cytolysin wherein highly conservedcysteine residues (e.g., cysteine 460 in PFO, cysteine 486 in LLO) arereplaced by conservative amino acid substitutions which are not subjectto reduction in order to prepare oxidation/reduction-insensitivecytolysin mutants which exhibit improved lytic activity. Alternatively,mutant cytolysins are selected from naturally occurring mutants by, forexample, identifying bacteria which contain cytolysins that are capableof lysing cells over a narrow pH range, preferably the pH range whichoccurs in phagosomes (pH 5.0-6.0), or under other conditions (e.g.,ionic strength) which occur in the targeted phagosomes. Nonsecretedcytolysins may be provided by various mechanisms, e.g. absence of afunctional signal sequence, a secretion incompetent microbe, such asmicrobes having genetic lesions (e.g. a functional signal sequencemutation), or poisoned microbes, etc.

The bacteria also comprise a second gene encoding a foreign agentdifferent from the cytolysin, and the subject methods may be used todeliver a wide variety of such foreign agents for a variety ofapplications, including diagnosis, therapy including prophylactics suchas immunizations (see, e.g. HIV vaccine, Table 1) and treatments such asgene therapy (especially of single gene disorders amenable to localizedtreatment, see Table 1, below), biosynthesis, etc.; essentially anyagent that the microbial host can be engineered to produce. In aparticular embodiment, the agent is largely retained by the microbeuntil lysis within the target cell vacuole. Note that the first andsecond genes may be the same, i.e. the same nucleic acid encodes boththe cytolysin and the foreign agent. For example, in a particularembodiment, the foreign agent is expressed in frame with the cytolysinas a fusion protein. In other embodiments, the microbes are engineeredto deliver libraries of agents for screening, e.g. Tenson T, et al., JBiol Chem 1997 Jul 11;272(28):17425-17430.

A wide variety of nucleic acid-based agents may be delivered, includingexpression vectors, probes, primers, antisense nucleic acids,knockout/in vectors, ribozymes, etc. For example, the subject bacteriaare used to deliver nucleic acids which provide templates fortranscription or translation or provide modulators of transcriptionand/or translation by hybridizing to selected endogenous templates, see,e.g. U.S. Pat. No. 5,399,346 for a non-limiting list of genes that canbe administered using gene therapy and diseases that can be treated bygene therapy. For example, polynucleotide agents may provide a codingregion operably linked to a transcriptional regulatory region functionalin a target mammalian cell, e.g. a human cytomegalovirus (CMV) promoter.In particular, the polynucleotide may encode a transcription factor,whereby expression of the transcription factor in the target cellprovides activation or de-activation of targeted gene expression in thetarget cell. In another example, RNA virus infected cells are targetedby microbes delivering viral RNA-specific ribozymes, e.g. HIV-infectedT-cells, leukemia virus infected leukocytes, hepatitis C infected livercells. In yet another embodiment, labeled probes are delivered whicheffect in situ hybridization-based diagnostics.

A wide variety of polypeptide-based agents may also be delivered,including antibiotics, insecticides, fungicides, anti-viral agents,anti-protozoan agents, enzymes, anti-cancer agents (e.g. cyclindependent kinase (CDK) inhibitors such as P16, P21 or P27), antibodies,anti-inflammatory peptides, transcription factors, antigenic peptides,etc. Exemplary therapeutically active polypeptides which can bedelivered by the subject invention are described in Nature Biotech16(2), entire issue, etc. In a particular embodiment, the inventionprovides for the delivery to antigen-presenting cells of antigenicpolypeptides which are presented in association with MHC proteins. Inanother particular embodiment, both nucleic acids and proteins aredelivered together contemporaneously, in the same administration or inthe same microbe. In some such applications, the nucleic acids andproteins can act in concert, e.g. an integrating vector and anintegrase, and RNA and a reverse transcriptase, a transposon and atransposase, etc.

The subject methods may also be used to deliver a wide variety of otherforeign agents that are synthesized by the host microbe. For example,microbes may be selected for, or engineered to contain, biosyntheticmachinery to produce any microbiologically producible agent compatiblewith the subject methods (e.g. sufficiently microbe impermeant toprovide effective delivery to the target cell). Preferred such agentsare those that are contraindicated for convenient direct (e.g. oral)administration, because of, for example, gut inactivation, toxicity,intolerance, impermeability, etc. In fact, even agents providingsignificant toxicity to the microbial host find use so long as aneffective amount of the agent may be loaded (by synthesis) or maintainedin the microbe (see, e.g. LLO toxicity, below).

A wide variety of nonvirulent, non-pathogenic bacteria may be used;preferred microbes are relatively well characterized strains,particularly laboratory strains of E. coli, such as MC4100, MC1061,DH5α, etc. Other bacteria that can be engineered for the inventioninclude well-characterized, nonvirulent, non-pathogenic strains ofListeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella,Bacillus subtilis, etc. In a particular embodiment, the bacteria areattenuated to be nonreplicative, nonintegrative into the host cellgenome, and/or non-motile inter- or intra-cellularly. A wide variety ofsuitable means for microbial attenuation are known in the art. Inanother particular embodiment, the bacteria are dead or non-viable priorto endocytosis by the target cell or administration to the targetorganism, obviating any microbial growth or metabolism in the targetcell. A wide variety of suitable means for killing or rendering thebacteria nonviable are known in the art, including fixation with organicsolvents such as methanol, UV irradiation, heat, freeze-drying, etc.Preferred methods preserve the ability of the microbial membrane and/orwall to retain the cytolysin and the foreign agent. In this embodiment,the first and second genes are sufficiently expressed to load themicrobe with an effective amount of the cytolysin and foreign agentprior to microbial cell death. Generally the bacteria contain (i.e. areloaded by expression within the bacteria with) with from about ten toone thousand, preferably from about one hundred to one thousandcytolysin molecules per bacterium.

The microbes of the invention can be used to deliver the foreign agentto virtually any target cell capable of endocytosis of the subjectmicrobe, including phagocytic, non-phagocytic, pathogenic or diseasedcells. Exemplary target animal cells include epithelial cells,endothelial cells, muscle cells, liver cells, pancreatic cells, neuralcells, fibroblasts, tumor cells, leukocytes such as macrophages,neutrophils, B-cells, T-cells, monocytes, etc., etc. The subject methodsgenerally require microbial uptake by the target cell and subsequentlysis within the target cell vacuole (including phagosomes andendosomes). While phagocytic target cells generally provide formicrobial uptake and lysis, for many cell types, it is necessary toprovide the bacteria with an invasin to facilitate or mediate uptake bythe target cell and an autolysin to facilitate or mediate autolysis ofthe bacteria within the target cell vacuole. A wide variety of suitableinvasins and autolysins are known in the art. For example, both Sizemoreet al. (Science, 1995, 270:299-302) and Courvalin et al. (C. R. Acad.Sci. Paris, 1995, 318: 1207-12) teach expression of an invasin to effectendocytosis of the bacterium by a target cell and suitable microbialautolysins are described by Cao et al., Infect Immun 1998, 66(6):2984-2986; Margot et al., J. Bacteriol 1998, 180(3):749-752; Buist etal., Appl Environ Microbiol, 1997, 63(7):2722-2728; Yamanaka et al.,FEMS Microbiol Lett, 1997, 150(2): 269-275; Romero et al., FEMSMicrobiol Lett, 1993, 108(1):87-92; Betzner and Keck, Mol Gen Genet,1989, 219(3): 489-491; Lubitz et al., J. Bacteriol, 1984,159(1):385-387; and Tomasz et al., J. Bacteriol, 1988, 170(12):5931-5934. Providing the advantage of delayed lysis aretemperature-sensitive autolysins, time-sensitive autolysins (see, e.g.Chang et al., 1995, J Bact 177, 3283-3294; Raab et al., 1985, J Mol Biol19, 95-105; Gerds et al., 1995, Mol Microbiol 17, 205-210) and addiction(poison/antidote) autolysins, (see e.g. Magnuson R., et al., 1996, JBiol Chem. 271(31), 18705-18710; Smith A S, et al., 1997, Mol Microbiol.26(5), 961-970).

Administration of the microbe to target cells may be in vitro or in vivoaccording to conventional methodologies. In either case, the methodsgenerally involve growing the microbes, inducing the expression of thefirst and second genes, and contacting the target cells with aneffective amount of bacteria sufficient to effect the desired activityof the foreign agent in the target cell. Immunofluorescense may be usedto image and track the contents of the bacteria upon administration tothe cells in vivo or in vitro.

In vitro or ex vivo administration generally involves contacting thetarget cell with an effective amount of the microbes of the invention.Exemplary in vitro administrations are described and/or cited byreference below. In vitro applications include protein delivery (e.g.for functional determinations, toxin delivery to targeted cells inculture, half-life, degradation and localization determinations),nucleic acid delivery (e.g. DNA to transfected cell lines, genomiclibraries to screen and identify specific antigens, i.e. expressioncloning, etc.)

In vivo administration generally involves administering a pharmaceuticalcomposition containing a therapeutically effective amount of themicrobes of the invention. Generally, the therapeutically effectiveamount is between about 1 μg and 100 mg/kg, preferably between about 1μg and 1 mg/kg. The microbes are formulated into a pharmaceuticalcomposition by combination with an appropriate pharmaceuticallyacceptable excipient in accordance with routine procedures known to oneof ordinary skill in the art. The microbes may be used alone or inappropriate association, as well as in combination with otherpharmaceutically active compounds. The microbes may be formulated intopreparations in solid, semisolid, or liquid form such as tablets,capsules, powders, granules, ointments, solutions, suppositories, andinjections, in usual ways for topical, nasal, oral, parenteral, orsurgical administration. Administration in vivo can be oral, mucosal,nasal, bronchial, parenteral, subcutaneous, intravenous, intra-arterial,intra-muscular, intra-organ, intra-tumoral, surgical or in general byany means typical of a gene therapy administration. Administration willbe selected as is appropriate for the targeted host cells. Target cellsmay also be removed from the subject, treated ex vivo, and the cellsthen replaced into the subject. Exemplary methods for in vivoadministration are described in Shen et al., Proc Natl Acad Sci USA1995, 92(9):3987-3991; Jensen et al, Immunol Rev 1997, 158: 147-157;Szalay et al., Proc Natl Acad Sci USA 1995, 92(26):12389-12392; Belyi etal, FEMS Immunol Med Microbiol 1996, 13(3): 211-213; Frankel et al., J.Immunol 1995, 155(10):4775-4782; Goossens et al., Int Immunol 1995,7(5):797-805; Schafer et al., J. Immunol 1992, 149(1):53-59; and Lindeet al., Vaccine 1991, 9(2):101-105.

The foregoing methods and compositions are demonstrated to be effectivein a wide variety of exemplary applications. In one application, a K12strain of E.coli is engineered with a signal sequence deficient LLO geneoperably linked to the constitutive tet promoter for expressing thecytolysin in the bacterium and a second gene encoding a truncated BRCA1cancer antigen, under regulatory control of a trc or tac promoter. Thecytolysin and cancer antigen are expressed to maximum levels, thebacteria are then fixed with methanol, and the killed bacteria loadedwith the cytolysin and cancer antigen are then injected into solidbreast tumors in three weekly injections. At four weeks, a cancerantigen-specific cytotoxic T-cell response (CTL response) and tumor sizereduction is detected. As shown in Table 1, analogous studies conductedin a variety of animals and animal cell types, both in vivo and invitro, using a variety of agents, secretory deficient cytolysins,bacterial types and methods demonstrate consistent delivery of the agentto the target cell cytosol, as measured by agent activity, immunoassay,or other delivery monitoring assays described herein.

TABLE 1 Microbial-Based Delivery Cystosol Target Cell Indication AgentLysin Bacteria Administration Delivery transformed acites tumor in nudehuman tumor antigen hTA1 LLO E.coli, JM109 (DE3) intraperitoneal +++human mice injection macrophage rat liver hepatocellular p51 tumorsuppressor LLO^(M1) E.coli, DP-E3619, in situ; intratumor +++ carcinomainvasin/autolysin injection rat kidney genetic nephopathy angiotensinconverting enzyme LLO^(M2) S. typhimurium, attenuated, ex vivo +++invasin/autolysin mouse brain neurodegeneration cFos gene expressionconstruct LLO^(M3) S. typhimurium, attenuated, in situ; intracranial +++invasin/autolysin implant mouse pancreas transformation γ-interferonexpression construct LLO^(M4) E.coli, JM109 (DE3) in vitro +++ pigmuscle muscular atrophy insulin-like growth factor I (IGF-I) PLO E.coli, DP-E3618, in situ; i.m. +++ invasin/autolysin injection humanbreast transformation anti-estrogen receptor antibody SLO E. coli,DP-3617, in vitro +++ expression construct invasin/autolysin humanprostate localized prostatic ribozyme or antisense against CDK LLO S.typhimurium, attenuated in situ; intratumor +++ carcinoma 2 or CDK4invasin/autolysin injection human lymphoid lymphoma tumor necrosisfactor (TNF) LLO E. coli, DP-E3616 ex vivo +++ human bone mylomoidleukemia Hepatocyte growth factor/scatter SLO E. coli, DP-E3615 ex vivo+++ marrow factor (HGF/SF) human lymphoid HIV infection HIV RTgene-specific ribozyme LLO S. typhimurium, attenuated ex vivo +++ humanhepatic Hepatitis C infection Hepatitis C virus-specific ribozyme LLO S.typhimurium, attenuated, in vivo; direct +++ cells invasin/autolysininjection human hepatic diabetes insulin receptor expression LLO S.typhimurium, attenuated, in vivo; direct +++ cells constructinvasin/autolysin injection human beta islet diabetes insulin expressionconstruct LLO S. typhimurium, attenuated, in vivo; direct +++ cellsinvasin/autolysin injection murine fibroblast fibroblastoma diptheriatoxin LLO S. typhimurium, attenuated, in vivo; direct +++invasin/autolysin injection feline retinal retinal degenerative cGMPphosphodiesterase-beta LLO E. coli, JM109 (DE3), intraocular +++ cellsdisease invasin/autolysin injection human cytotoxic melanoma melanosomalproteins LLO E.coli, JM109 (DE3) in vivo; IV +++ T-cells injection humanepithelium Herpes infection antisense RNAseP construct LLO S.typhimurium, attenuated, in vivo; oral, opical +++ invasin/autolysinabrasion murine IL-2 production NFAT LLO S. typhimurium in vitro +++macrophages LLO^(M1-M4) are LLO mutants M1(Cys486Ser), M2(Cys486Met),M3(Trp492Ala) and M4(del491-493), respectively.

EXAMPLES

I. Delivery of Protein to the Cytosol of Macrophages Using Escherichiacoli K-12 Expressing Listeriolysin O

Listeria monocytogenes is a bacterial pathogen that replicates withinthe cytosol of mammalian cells. L. monocytogenes has been usedextensively as a model for the study of cell-mediated immunity and as amodel pathogen for understanding the basis of intracellular pathogenesis(13, 14). Following internalization into host cells, bacteria areinitially contained within host vacuoles then subsequently lyse thesevacuoles to gain access to the cytosol. The ability of L. monocytogenesto lyse the vacuole and enter the cytosol is primarily mediated bylisteriolysin O (LLO). LLO is a member of a family of relatedpore-forming cytolysins secreted by diverse species of gram positivebacteria (15). LLO encapsulated into pH-sensitive liposomes has beenused as a vehicle to deliver co-encapsulated protein to the cytosol ofmacrophages (16). Moreover, purified LLO when mixed with foreignproteins and added to mammalian cells can mediate the delivery ofprotein to the cytosol and has been exploited for delivery to host cellsboth in vitro and in vivo (17-19). However, both of these methodsrequire the purification of LLO and the protein to be delivered. Here,we show that Escherichia coli expressing cytoplasmic LLO can be used toefficiently deliver co-expressed proteins to the cytosol of macrophages.The utility of this system to deliver a large active protein to thecytosol was demonstrated by the delivery of E. coli β-galactosidase(β-gal). Using chicken ovalbumin (OVA) we demonstrate the rapid deliveryof protein to the cytosol of macrophages and the ability of the E.coli/LLO system to efficiently deliver OVA to the MHC class I pathway ofantigen processing and presentation. Moreover, the time required forprocessing and presentation of an OVA-derived peptide to CD8⁺T cells,when OVA was delivered using this system, was equivalent to thatpreviously reported when purified OVA was introduced into the cytosol byalternative methods such as scrape-loading or liposomes (16, 20, 21).

Bacterial Strains and Plasmids. All bacterial strains and plasmids usedin this report are listed in Table 2.

TABLE 2 E. coli strains and plasmids used in this work Strain or PlasmidDescription Reference or source pACYC184 cloning vector; Tc^(r) Cm^(r)(23) pET28a over-expression vector; Kan^(r) (Novagen, Inc.) pET29bover-expression vector; Kan^(r) (Novagen, Inc.) pTL61T lacZtranscriptional fusion vector; Ap^(r) (24) pBluescript SK- cloningvector; Ap^(r) (Stratagene, Inc.) pTrcHisC/Ova pTrcHisC::ova (D.Campbell, UCB) pDP3615 pACYC184 tet::hly Herein pDP3616 pET28a pT7::ovaHerein pDH70 pBluescript SK- pT7::lacZ Herein MC4100(DE3) F⁻ araD 139Δ(argF-lac)U169 rpsL150 (Schifferli, U. Penn) (Str^(r))relAl flbB530IdeoCl ptsF25 rbsR with DE3, a λ prophage carrying the T7 RNA polymerasegene JM109(DE3) endAl recAl gyrA96 thi hsdR17 relAl supE44 (Promega,Co.) Δ(lac-proAB) [F′ traD36 proAB lacl^(q)ZΔM15] DE3 DP-E3615MC4100(DE3) harboring pDP3615 Herein DP-E3616 MC4100(DE3) harboringpDP3616 Herein DP-E3617 MC4100(DE3) harboring pDP3615 and pDP3616 HereinDP-E3618 JM109(DE3) harboring pDH70 Herein DP-E3619 JM1O9(DE3) harboringpDP3615 and pDH70 Herein Ap^(r), ampicillin resistant; Tc^(r),tetracycline resistant; Kan^(r), kanamycin resistant; Cm^(r),chloramphenicol resistant; Str^(r), streptomycin resistant

Plasmid pDP3615 was generated by PCR amplification of the hly geneencoding LLO lacking its secretion signal sequence (22). DNA sequencesencoding mature LLO were first PCR amplified and cloned into pET29b(Novagen, Inc., Madison, Wis.) using oligonucleotide primer5′-GGAATTCCATATGAAGGATGCATCTGCATTCAAT-3′ (SEQ ID NO:1) generating a NdeIrestriction site at the 5′ end of the gene fragment and primer5′-CGGGATCCTTATTATTCGATTGGATTATCTACT-3′ (SEQ ID NO:2) generating a BamHIrestriction site at the 3′ end of the gene fragment. Following ligationinto the pET29b vector, the DNA sequences encoding mature LLO along withthe upstream translation initiation site found in pET29b were amplifiedusing primer 5′-CGCGATATCCTCTAGAAATAATTTTG-3′ (SEQ ID NO:3) generatingan EcoRI restriction site at the 5′ end of the gene fragment and thesame primer used previously to generate a BamHI restriction site at the3′ end of the gene fragment. The amplified fragment was ligated intopACYC 184 (23) placing transcription of the mature hly gene undercontrol of the tet gene promoter. Plasmid pDP3616 was generated bysubcloning a NcoI-HindIII fragment containing DNA sequences encodingtruncated OVA from plasmid pTrcHisC/OVA. The DNA fragment was ligatedinto the over-expression vector pET28a (Novagen, Inc., Madison, Wis.).Plasmid pDH70 was generated by PCR amplification of the promoterlesslacZ gene in plasmid pTL61T (24) using oligonucleotide primers5′-AGGCGTCGACGGTTAATACGACCGGGATCGAG-3′ (SEQ ID NO:4) and5′-AGGCGTCGACAGGCCTTACGCGAAATACGGGCAGACATGG-3′ (SEQ ID NO:5) generatingSalI restriction sites at both the 5′ and 3′ ends of the fragment. Theamplified fragment was ligated into pBluescript SK-(Stratagene, Inc., LaJolla, Calif.) placing transcription of the lacZ gene under control of aphage T7 promoter. Plasmid DNA was transferred to E. coli strains bytransformation, using standard methods (25). E. coli strains were grownin Luria-Bertani (LB) medium. The strains were stored at −70° C. in LBmedium plus 40% glycerol. Antibiotics were used at the followingconcentrations: ampicillin, 100 μg/ml; chloramphenicol, 40 μg/ml; andkanamycin, 30 μg/ml.

Expression of Target Proteins. E. coli strains were inoculated from a LBagar plate into 2 mls of LB medium and grown overnight to stationaryphase at 37° C. with aeration. Cultures were diluted 1:100 in 10 mls ofLB medium in 250 ml flasks and grown 2 hours with aeration at 30° C.Target protein expression was induced by the addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to 1 mM and growth continueduntil cultures reached an OD₆₀₀ of 0.5. Equivalent numbers of bacteriawere centrifuged (14,000×g) for 1 minute and washed once with phosphatebuffered saline (PBS). Washed samples were suspended in Final SampleBuffer (0.0625M Tris pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenolblue) boiled for 5 minutes and total cellular protein analyzed bypolyacrylamide gel electrophoresis followed by staining with CoomassieBrilliant Blue.

Determination of Hemolytic Activity. Following bacterial growth andinduction of target proteins, 1 ml aliquots of bacteria were centrifuged(14,000×g) for 1 minute and washed once with PBS. Samples wereresuspended in 1 ml of PBS and lysed by sonication. Soluble extractfractions were obtained by centrifuging lysed samples for 10 minutes(14,000×g) at 4° C. and saving the supernatant. Hemolytic activity inthe soluble fractions was determined as previously described (26) and isexpressed as the reciprocal of the dilution of extracts required to lyse50% of sheep erythrocytes.

Cell Culture. Cell lines were maintained in RPMI 1640 medium or DMEMsupplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc.,Logan, Utah), 2 mM glutamine, 1 mM pyruvate, 50 μM 2-mercaptoethanol,penicillin (200 units/ml), and streptomycin (200 μg/ml) at 37° C. in a5% CO₂/air atmosphere. The IC-21 and Raw 309 Cr.1 mouse macrophage celllines were obtained from the American Type Culture Collection (ATCC,Rockford, Md.). The B3Z T-cell hybrid is a LacZ-inducible CD8⁺ T-cellhybridoma specific for OVA residues 257-264, SIINFEKL (SL8)(SEQ IDNO:6), presented on the murine K^(b) MHC class I molecule (27, 28).

Delivery of Protein to the Cytosol of Macrophages. IC-21 cells wereseeded onto 18 mm glass coverslips in 35 mm dishes in RPMI mediumwithout antibiotics. One hour prior to addition of bacteria, dishes wereplaced at 4° C. Medium was removed from the dishes and E. coli wereadded in cold RPMI medium without antibiotics to obtain an infectionratio of one bacterium/macrophage. Samples were incubated at 4° C. forone hour to allow association of bacteria and macrophages. Samples werewashed five times with 3 mls of cold PBS and 37° C. RPMI medium added.Samples were incubated at 37° C. in a 5% CO₂/air atmosphere for up toone hour. At varying intervals during incubation of macrophages and E.coli, coverslips were removed and fixed for subsequent detection ofprotein.

Detection of Target Protein in Macrophages. Following delivery of β-galto macrophages, coverslips were fixed in cold 2% formaldehyde/0.2%glutaraldehyde for 5 minutes at 4° C. β-gal activity was detected bystaining with 5-bromo-4-chloro-3-indolyl β-galactopyranoside (X-gal,Sigma Immunochemicals, St. Louis, Mo.) as previously described (27). Fordetection of OVA, coverslips were fixed in 3.2% electron microscopygrade paraformaldehyde (Electron Microscopy Sciences, Ft. Washington,Pa.) overnight at 4° C. in aluminum foil wrapped containers. OVA wasdetected by immunofluorescence as previously described (29) with theexception that polyclonal rabbit anti-OVA antibody (Calbiochem, SanDiego, Calif.) and LRSC-conjugated donkey anti-rabbit IgG antibody(Jackson ImmunoResearch, West Grove, Pa.) were used for detection.

Antigen Presentation Assays. Presentation of SL8/K^(b) complex to B3Zcells was determined as previously described (30). Briefly, E. coli wereadded to 1×10⁵ antigen presenting cells (APCs) in each well of a 96-wellmicrotiter plate. Following one hour of incubation at 37° C. in a 5%CO₂/air atmosphere, extracellular bacteria were removed by washing threetimes with PBS and 1×10⁵ B3Z T cells were added to each well in mediumcontaining 100 μg/ml gentamicin. Following 15 hours of incubation at 37°C. in a 5% CO₂/air atmosphere, cultures were washed once with PBS andlysed by addition of 100 μl PBS buffer containing 100 μM2-mercaptoethanol, 9 mM MgCl₂, 0.125% NP40, and 0.15 mMchlorophenolred-β-galactoside (CPRG, Calbiochem, San Diego, Calif.).After 4-6 hours at 37° C., 50 μl of stop buffer (300 mM glycine and 15mM EDTA in water) was added and the absorbance at 570 nm of each wellwas determined using a 96-well plate reader. Where appropriate, APCswere fixed with 1% paraformaldehyde prior to the addition of B3Z cellsas described (31). Synthetic SL8 peptide was obtained from ResearchGenetics, Inc. (Huntsville, Ala.) and was incubated with APCs and B3Zcells at a saturating concentration of 90 nM in RPMI medium.

Expression of Listeriolysin O and Target Proteins. To facilitateexpression of mature cytoplasmic LLO in E. coli, the hly gene encodingLLO lacking its N-terminal signal sequence (22) was cloned into theplasmid vector pACYC184 to generate pDP3615 as described in Materialsand Methods. Transcription of the truncated hly gene in pDP3615 is underthe control of the constitutive tet gene promoter. Proteins to bedelivered to the cytosol of macrophages were expressed from co-residentplasmids in E. coli. We chose chicken ovalbumin (OVA) as one of therepresentative proteins to deliver to the cytosol of macrophages. OVA isnot toxic to E. coli and can be readily expressed to high levels (32). Aplasmid encoding truncated (32 kD) OVA was transformed into E. colialong with pDP3615. In order to determine if a large protein with ameasurable enzymatic activity could be delivered to the cytosol ofmacrophages, we expressed β-galactosidase (β-gal) along with LLO in E.coli. A plasmid containing the gene encoding β-gal, was transformed intoE. coli along with plasmid pDP3615. Expression of both OVA and β-gal inthese strains is under the control of IPTG-inducible phage T7 RNApolymerase. We next analyzed the hemolytic activity and proteinexpression profiles of these strains. Following IPTG induction, OVA andβ-gal were expressed to approximately 20% of the total E. coli cellularprotein as determined by SDS-PAGE. To verify expression of active LLOprotein within E. coli, hemolytic activity contained in the solublefraction of E. Coli extracts was determined as described above. All ofthe strains expressing LLO contained approximately 500-600 hemolyticunits of activity in the soluble extracts. No measurable hemolyticactivity was found in the culture medium in which the E. coli weregrown. These data indicate that functional LLO protein was containedwithin the E. coli cells and not secreted to the extracellularenvironment.

Delivery of Protein to the Cytosol of Macrophages. To examine theability of E. Coli expressing LLO to deliver a co-expressed targetprotein to the cytosol of macrophages, E. coli expressing LLO and OVAwere added to macrophages to obtain an infection ratio of approximatelyone bacterium/macrophage. The presence of OVA either in phagosomes or inthe cytosol was determined by immunofluorescence microscopy. Whenbacteria expressing OVA in the absence of LLO were added to macrophages,protein was contained within phagosomes and no OVA could be detected inthe cytosol of macrophages within one hour following phagocytosis. Incontrast, when bacteria expressing both LLO and OVA were added tomacrophages, OVA protein could be detected throughout the entirecytosolic compartment within 30 minutes of bacterial uptake. Moreover,at least 50% of macrophages that had phagocytosed a single bacteriumdemonstrated release of OVA into the cytosol within 30 minutes followingbacterial uptake. In some instances, OVA protein could be detectedleaking from phagosomes into the cytosol as early as 10 minutespost-phagocytosis of bacteria.

Since release of OVA into the cytosol occurs subsequent to degradationof the E. coli within macrophage phagosomes, it was possible thatproteins contained within the bacteria were also partially degraded orinactivated before release into the cytosol. To examine whetherfull-length β-gal could be delivered to the cytosol of macrophages andretain its biological activity, E. coli expressing LLO and β-gal wereadded to macrophages to obtain an infection ratio of approximately onebacterium/macrophage. β-gal activity in the cytosol was then determinedby staining macrophages with X-gal. Our data indicate that β-galactivity could be detected throughout the cytosol within 30 minutesfollowing bacterial uptake. Following phagocytosis of E. coli expressingβ-gal in the absence of LLO, β-gal activity was detected sequesteredwithin phagosomes. β-gal is a 116 kD protein that functions as a 465 kDtetramer (33). Whether β-gal is associated as a tetramer prior torelease into the cytosol is unknown, but these data indicate that at aminimum a 116 kD protein can be delivered to the cytosol of macrophagesusing the E. coli/LLO delivery system and still retain its enzymaticactivity.

Delivery of OVA to the MHC Class I Pathway for Antigen Presentation.Immunity to intracellular bacterial pathogens and viruses often requiresthe generation of cytotoxic T-lymphocytes (CTLs) that recognize and killinfected cells (13, 34, 35). Efficient processing and presentation ofantigens to CTLs typically requires the endogenous synthesis of theantigen within the cytosol of the infected cell or introduction of theantigenic protein into the cytosol of an antigen presenting cell (APC)(36, 37). Once in the cytosol, proteases process the antigen to peptideepitopes which are subsequently presented on the surface of the APC inassociation with major histocompatibility (MHC) class I molecules forrecognition by CD8⁺ CTLs (37-39).

We wished to examine the ability of E. coli expressing LLO and anantigenic protein to deliver the antigen to the cytosol of macrophagesfor processing and presentation on MHC class I molecules. E. coliexpressing LLO and OVA were added to macrophages and the processing andpresentation of a peptide epitope derived from OVA was accessed usingthe B3Z T-cell hybrid. B3Z is a LacZ-inducible CD8⁺ T-cell hybridspecific for OVA residues 257-264 (SIINFEKL, SEQ ID NO:6) presented onthe murine K^(b) MHC class I molecule (27, 28). The presentation of theSIINFEKL (SEQ ID NO:6) epitope (SL8) to B3Z cells results in theinduction of β-gal synthesis by B3Z. The amount of β-gal produced can bemeasured by the hydrolysis of the chromogenic substrate CPRG and is anindication of the amount of SL8/K^(b) complexes presented on the surfaceof APCs (27, 30). Bacteria were added to macrophages and phagocytosisallowed to proceed for one hour, followed by the removal ofextracellular bacteria and addition of B3Z T-cells. As shown in FIG. 1,processing and presentation of SL8/K^(b) to B3Z T-cells occurred when E.coli expressing both LLO and OVA were added to macrophages. In thisfigure, the indicated number of E. coli were added to 1×10⁵ Raw 309 Cr.1APCs in each well of a 96-well plate. Following one hour of incubationat 37° C. in a 5% CO₂/air atmosphere, extracellular bacteria wereremoved by washing with PBS and 1×10⁵ B3Z T-cells were added to eachwell in medium containing 100 μg/ml gentamicin. Following 15 hours ofincubation at 37° C. in a 5% CO₂/air atmosphere, presentation ofSL8/K^(b) to B3Z cells was assayed as described (27, 30) and indicatedas an increase in the absorbance at 570 nm. E. coli strains added toAPCs expressed LLO, DP-E3615 (∘); OVA, DP-E3616 (); or LLO and OVA,DP-E3617, (□). (▪) indicates the level of activation obtained when APCswere incubated with 90 nM synthetic SL8 and B3Z cells. Data presented isfrom triplicate groups of wells from one of several repeated experimentswith identical results. As shown, antigen presentation could be detectedwith as few as 1 bacterium added/10 macrophages. At a ratio of 10bacteria added/macrophage, which resulted in the phagocytosis of one totwo bacteria/macrophage, the level of presentation was equivalent to themaximal activity achieved by incubating macrophages with a saturatingdose (90 nM) of synthetic SL8 peptide. No presentation of SL8/K^(b)complex could be detected when E. coli expressing OVA in the absence ofLLO were added to macrophages. Equivalent results were obtained whenprimary bone marrow and peritoneal derived macrophages were used inantigen presentation experiments. The decrease in absorbance seen when10⁷ and 10⁸ LLO expressing bacteria were added was due to visible damageto the macrophages.

Processing and Presentation of SL8/K^(b) Complex is Rapid. It has beenpreviously demonstrated that OVA is efficiently processed andpeptide/MHC complexes presented on the surface of APCs within two tofour hours following delivery of OVA to the cytosol using alternativemethods such as encapsulation in liposomes or scrape-loading (16, 20,21). Data in FIG. 1 indicate that considerable processing andpresentation of antigenic peptides can occur with as few as onebacterium added/macrophage. However, processing and presentation ofantigen was allowed to occur for greater than 15 hours prior tomeasuring T-cell activation. It is possible that the time required forefficient processing of antigen has been altered by delivering proteinto the cytosol using this method. We wished to examine the timenecessary for antigen processing and presentation of peptide/MHCcomplexes when protein is delivered using the E. coli/LLO system.

Paraformaldehyde fixation of macrophages prevents further phagocytosisof bacteria and has been shown to crosslink surface MHC class Imolecules to associated β₂-microglobulin (31). Thus, fixation stabilizespeptide/MHC complexes present on the cell surface and arrests anyfurther processing and presentation of peptides. We examined the timerequired for antigen processing and presentation by fixing APCs withparaformaldehyde at varying intervals after addition of bacteria andmeasuring SL8/K^(b) presentation to B3Z T-cells. First, the effect onantigen presentation of fixing macrophages prior to the addition ofbacteria was addressed (FIG. 2A). In FIG. 2A, 1×10⁶ E. coli strainDP-E3617 expressing LLO and OVA were added to 1×10⁵ IC-21 macrophages ineach well of a 96-well plate. Processing and presentation of SL8/K^(b)was assayed as described in FIG. 1. Immediately prior to the addition ofbacteria, APCs were either left untreated (Not Fixed) or fixed(Pre-Fixed) with 1% paraformaldehyde as described (31) Fixing APCs priorto the addition of bacteria completely abrogated the ability ofmacrophages to process and present SL8/K^(b) complex to B3Z T-cells, asevident by an equivalent response as that seen when no bacteria wereadded to the macrophages (FIG. 1).

In FIG. 2B, the time requirement for processing and presentation ofSL8/K^(b) complex was addressed. Here, 1×10⁶ E. coli strain DP-E3617expressing LLO and OVA were added to 1×10⁵ IC-21 APCs in each well of a96-well plate. Following one hour of incubation at 37° C. in a 5%CO₂/air atmosphere, extracellular bacteria were removed by washing withPBS and APCs were either immediately fixed with 1% paraformaldehyde (1hour) or incubated further in media containing 100 μg/ml gentamicin. Atone hour intervals, APCs were fixed with 1% paraformaldehyde until alltime points were completed. Following completion of the four hour timeinterval, APCs were washed with PBS and 1×10⁵ B3Z T cells added to eachwell. Presentation of SL8/K^(b) was assayed as described above. Labelsindicate the time elapsed post addition of bacteria prior to fixation ofAPCs. Dark shaded bars indicate samples to which E. coli strain DP-E3617were added. Light shaded bar indicates samples to which no bacteria but90 nM synthetic SL8 was added with B3Z cells. The (Not Fixed) samplesreceived no fixation prior to the addition of B3Z cells. Data presentedis from triplicate groups of wells from one of three experiments withidentical results.

Fixing APCs at one hour following the addition of E. coli expressing LLOand OVA resulted in sufficient antigen presentation to yield activationof B3Z cells to a level slightly higher than those seen in the absenceof fixing (compare 1 hour fixed to Not Fixed). The increased level ofSL8/K^(b) presentation following fixation can be attributed tocrosslinking of surface MHC class I molecules resulting in increasedstability of peptide-MHC complexes (31). Consistent with previousstudies of OVA delivery to APCs (16, 20, 21), the maximal presentationof SL8/K^(b) complex occurred when processing of OVA was allowed tocontinue for two hours prior to fixation. This level of SL8/K^(b)presentation was equivalent to that seen with the addition of 90 nMsynthetic SL8 in the absence of fixation (FIG. 2B). Additional analysisindicated that fixing APCs later than two hours after addition ofbacteria, resulted in a decreased level of SL8/K^(b) presentation (FIG.2B, 3 and 4 hour time points). This is consistent with dissociation ofsurface peptide/MHC complexes prior to crosslinking MHC class Imolecules by fixation (31). These data indicate that no delay in theprocessing and presentation of SL8/K^(b) complex occurred when OVA wasdelivered to the cytosol using the E. coli/LLO delivery system.

The results of this example demonstrate that E. coli expressingcytoplasmic LLO can be used to deliver a co-expressed protein to thecytosol of macrophages. The delivery of protein to macrophages was rapidand efficient with protein first appearing in the cytosol within tenminutes following bacterial uptake. Moreover, large enzymatically activeproteins can be introduced into the cytosol using this method asdemonstrated by the delivery of active β-gal. The mechanism of deliverymay be as follows. Subsequent or concomitant to phagocytosis, the E.coli are killed and degraded within phagosomes causing the release ofLLO and the target protein from the bacteria. LLO acts by forming largepores in the phagosomal membrane thus releasing the target protein intothe cytosol. In any event, any protein that can be synthesized in E.coli can be delivered to the macrophage cytosol.

LLO is an essential determinant of pathogenesis whose role is to mediaterelease of L. monocytogenes from a phagosome. The biological propertiesof LLO make it well suited for use in our system. For example, LLO hasan acidic pH optimum which facilitates its action in a phagosome (40,41). However, it was unclear whether LLO released by degraded E. coliwould retain its biological activity. The data presented heredemonstrate that the amount of LLO expressed was sufficient to allow therapid release of protein into the cytosol. Based on SDS-PAGE analysis ofknown quantities of purified LLO protein, we estimate approximately1×10⁵ molecules of LLO per E. coli cell. Using our disclosure, one cannow determine how many molecules of LLO are actually needed to introducea pore into a phagosome as described (42), where it was determined totake only approximately 50 molecules of streptolysin O, a homologouspore-forming cytolysin, to form a pore in red cell membranes. A secondproperty of LLO is its relative lack of toxicity thought to be due toits proteolysis in the cytosol of host cells (43). Indeed, secretion byL. monocytogenes of a related pore-forming hemolysin, perfringolysin O,resulted in death of the infected cells (44). Other facultativeintracellular pathogens, Shigellae, Salmonellae, and Yersiniae allinduce macrophage apoptosis (14, 45), yet infection with L.monocytogenes is relatively benign (46). In the current study, eventhough we estimate each recombinant E. coli contained approximately1×10⁵ molecules of LLO, there was no evidence of toxicity until therewere about 25 bacteria/macrophage.

There are a number of advantages and applications of the E. coli/LLOdelivery system. Many methodologies for delivering protein to thecytosol of macrophages require the prior purification of the protein tobe delivered. With the E. coli/LLO mediated delivery of protein, noprotein purification is required, only expression of the target proteinin E. coli is necessary. Furthermore, with many alternative methods,delivery is restricted to minute amounts of protein or a limited numberof cells and the in vivo delivery of protein can not be achieved (2).Using the E. coli/LLO system, high levels of protein can be delivered tothe cytosol of virtually all of the cells in culture. In addition, byexpressing protein under the control of inducible promoters, the levelof protein produced and ultimately delivered to the cytosol ofmacrophages can be controlled. This system can be used in vivo and byexpressing invasive determinants from other bacterial species, the E.coli may be modified to enter cells other than macrophages. Furthermore,this system has applications for the delivery of pathogen-specificprotein antigens or DNA.

The results of this example show that the E. coli/LLO system isparticularly effective for the introduction of protein into the MHCclass I pathway of antigen processing and presentation. We were able todetect antigen presentation with less than 1 bacterium/10 macrophagesand observed a maximal response with as few as 1 to 2bacteria/macrophage (FIG. 1). In addition, efficient processing andsurface presentation of peptide/MHC complexes occurred rapidly, within1-2 hours following addition of bacteria to macrophages (FIG. 2B).Delivery to the MHC class I pathway was enhanced greater than 4-logscompared to E. coli expressing OVA alone. This is a similar level ofenhancement to that reported when OVA linked to beads was compared tosoluble OVA for presentation with MHC class I molecules (47). It isclear from subsequent studies that the beads, like LLO, mediateddisruption of the phagosome (21, 48). However, there was one report inwhich E. Coli expressing OVA was able to deliver OVA to the MHC class Ipathway (49). Here, delivery was proposed to occur by a non-conventionalpathway involving extracellular peptide regurgitation of phagosomalprocessed antigens instead of transfer of protein from the phagosomalcompartment to the cytosol. Nonetheless, our data clearly showundetectable levels of antigen presentation when E. coli lacking LLO yetexpressing OVA to 20% of the total cellular protein were used to deliverOVA to macrophages. Perhaps the T-cells used in our studies were unableto detect presentation of SL8/K^(b) complexes, when E. coli expressingOVA alone were used, because of inefficient processing and presentationvia the non-conventional pathway. In the previous study, OVA wasgenerated as fusions to Crl or LamB proteins. The efficiency ofprocessing and presentation of epitopes from OVA has been shown to bedependent on the protein context surrounding the epitope (50, 51).Therefore, it is possible that the fusion proteins used in the previousstudy are processed more efficiently than the truncated OVA used in thisreport.

Recently, an E. coli expression cloning strategy for the identificationof CD4⁻ T cell-stimulating antigens has been reported (30). However,this method has not been successfully used to identify CD8⁺CTL-stimulating antigens since proteins expressed in E. coli do not gainefficient access to the MHC class I pathway for antigen presentation.The results of this study indicate that the E. coli/LLO delivery systemprovides an expression cloning strategy for the identification ofpathogen-specific CD8⁺ CTL-stimulating antigens. The identification ofthese pathogen-specific epitopes is an important step in the rationaldesign of vaccine strategies against these infectious agents. Theseantigens are further characterized to determine the peptide epitopesrecognized by CTLs, as well as the natural function the antigenicprotein plays in the interaction of the pathogen and host cells.

The efficiency of antigen delivery provides for the E. coli/LLO systemto be used for the induction of CTLs in vivo. The efficient in vivodelivery of antigens to generate a protective immune response is asignificant challenge in vaccine development. The use of bacterialvectors that have evolved to invade and replicate in mammalian cellssuch as Shigella (11, 12), Salmonella (8-10), and Listeria monocytogenes(52-55) are being explored as methods for the delivery of both proteinantigens and DNA. Although these vehicles have had success in elicitingprotective immune responses, the in vivo use of pathogenic bacteria hasinherent risks. One strategy to overcome these obstacles has been toengineer attenuated E. coli that can invade and enter the cytosol ofhost cells for the delivery of macromolecules. E. coli deficient in theproduction of diaminopimelate (DAP), an essential cell wall component,undergo lysis during growth in the absence of DAP (56). DAP-minus E.coli carrying the 200 kb virulence plasmid pWR100 from Shigella flexnerihave been engineered to deliver DNA to mammalian cells (11). These E.coli have the ability to invade cultured cells and enter the cytosolsimilar to S. flexneri, yet following brief replication, spontaneouslylyse in the cytosol and allow for the delivery of DNA for subsequentexpression in the host cell. However, the presence of the pWR100virulence plasmid poses limitations on the suitability of this microbefor many applications. The rational design of safe delivery vectors istherefore of paramount importance when constructing new methodologiesfor in vivo delivery. Since the E. coli/LLO system does not contain anyvirulence associated determinants other than LLO, it is uniquelysituated to safely deliver antigens to macrophages in vivo to generate aprotective immune response.

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All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 34<212> TYPE: DNA <213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 1ggaattccat atgaaggatg catctgcatt caat        #                  #        34 <210> SEQ ID NO 2 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 2cgggatcctt attattcgat tggattatct act        #                  #         33 <210> SEQ ID NO 3 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 3cgcgatatcc tctagaaata attttg           #                  #              26 <210> SEQ ID NO 4 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 4aggcgtcgac ggttaatacg accgggatcg ag        #                  #          32 <210> SEQ ID NO 5 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 5aggcgtcgac aggccttacg cgaaatacgg gcagacatgg      #                  #    40 <210> SEQ ID NO 6 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: murine <400> SEQUENCE: 6 Ser Ile Ile Asn Phe Glu Lys Leu 1               5

What is claimed is:
 1. A method of introducing a foreign agent into aeukaryotic cell comprising the step of: contacting the cell with anonvirulent bacterium comprising a first gene encoding a nonsecretedforeign functional cytolysin operably linked to a heterologous promoterwhich expresses the cytolysin in the bacterium, and a second geneencoding a foreign therapeutic agent, different than the cytolysin,under conditions whereby the therapeutic agent enters the cell.
 2. Themethod of claim 1, wherein the bacterium is endocytosed into a vacuoleof the cell, the bacterium undergoes lysis and the cytolysin mediatestransfer of the therapeutic agent from the vacuole to the cytosol of thecell.
 3. The method of claim 1, wherein the bacterium is dead ornon-viable.
 4. The method of claim 1, wherein the bacterium comprisesthe cytolysin.
 5. The method of claim 1, wherein the therapeutic agentis synthesized by the bacterium.
 6. The method of claim 1, wherein thebacterium is nonreplicative and nonintegrative into the host cellgenome.
 7. The method of claim 1, wherein the bacterium is a dead ornonviable laboratory strain of E. coli.
 8. The method of claim 1,wherein the bacterium is a laboratory strain of E. coli and thebacterium comprises the cytolysin.
 9. The method of claim 1, wherein thebacterium is a dead or nonviable laboratory strain of E. coli and thebacterium comprises the cytolysin.
 10. The method of claim 1, whereinthe bacterium is a dead or nonviable laboratory strain of E. coli andthe bacterium comprises the cytolysin and the cytolysin islisteriolysin.
 11. The method of claim 1, wherein there is no growth ormetabolism of the bacterium in the eukaryotic cell.
 12. The method ofclaim 1, wherein the therapeutic agent is selected from an antibiotic,insecticide, fungicide, anti-viral agent, anti-protozoan agent, enzyme,anti-cancer agent, antibody, anti-inflammatory peptide, andtranscription factor.
 13. The method of claim 1, wherein the method isindicated by a disease selected from the group consisting of cancer,infection, degenerative disease, and diabetes.
 14. The method of claim1, wherein the cell is a leukocyte.
 15. The method of claim 1, whereinthe cell is a tumor cell.
 16. The method of claim 1, wherein thecontacting step comprises administering a pharmaceutical compositioncomprising a therapeutically effective amount of the nonvirulentbacterium.
 17. The method of claim 16, wherein the administration is invivo.
 18. The method of claim 16, wherein the administration is ex vivo.